STRUCTURE OF POLYTENE CHROMOSOME BANDS AND INTERBANDS

拟议的研究建立在最近对我们的硬件进行扩展的基础上， 软件，允许，在一个广义的分辨率重叠的方式，3- 亚细胞结构的三维重建， 和电子显微镜水平。最近的进展使收集成为可能 体内三维时间相关多波长图像 信息，并随后进行定量分析，以提供见解 染色体结构。 我们将此方法应用于高阶染色体的分析 结构和功能使用独特的间期多线染色体的 果蝇我们专注于细胞学鉴定的多线 染色体带，带间和蓬松（转录活性， 形态学上独特的染色体位点）。我们利用果蝇 变态，由昆虫激素蜕皮激素触发，作为一种方式， 靶向特征性转录级联。 这项建议的具体目标是： A）继续确定特定的化合物的三维结构， 多线染色体带、带间带和粉团的电镜观察 断层扫描 B）研究以下的体内结构动力学（定量运动分析）： 脉冲标记多线染色体内的DNA复制位点。 C）研究蜕皮激素诱导的喷流的体内结构动力学 形成和回归。 D）研究多线染色体膨胀的结构动力学， 相关蛋白（蜕皮激素受体和早期puff编码蛋白） 蜕皮激素反应。 E）使用成像共振能量转移方法，确定动力学 特定的蛋白质-蛋白质的关联。

The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3- dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. Recent progress makes possible collection of in vivo 3-dimensional time dependent multiple wavelength image information, with subsequent quantitative analysis, to provide insights into chromosome structure. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase polytene chromosomes of Drosophila melanogaster. We focus on cytologically identified polytene chromosome bands, interbands and puffs (transcriptionally active, morphologically distinctive chromosome sites). We utilize Drosophila metamorphosis, triggered by the insect hormone ecdysone, as a way to target a characteristic transcriptional cascade. The specific aims of this proposal are to: A) Continue the determination of the 3-dimensional structure of specific polytene chromosome bands, interbands and puffs using electron microscope tomography. B) Study the in vivo structural dynamics (quantitative motion analysis) of pulse labeled DNA replication sites within polytene chromosomes. C) Study the in vivo structural dynamics of ecdysone induced puff formation and regression. D) Study the structural dynamics, on polytene chromosome puffs, of proteins (ecdysone receptor and early puff encoded proteins) associated with the ecdysone response. E) Using imaging resonance energy transfer methods, determine the dynamics of specific protein-protein associations within puff structures.